Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy

The organization of collagen during fibrotic processes is poorly characterized because of the lack of appropriate methodologies. Here we show that multimodal multiphoton microscopy provides novel insights into lung fibrosis. We characterize normal and fibrotic pulmonary tissue in the bleomycin model, and show that second-harmonic generation by fibrillar collagen reveals the micrometer-scale three-dimensional spatial distribution of the fibrosis. We find that combined two-photon excited fluorescence and second-harmonic imaging of unstained lung tissue allows separating the inflammatory and fibrotic steps in this pathology, underlining characteristic features of fibroblastic foci in human Idiopathic Pulmonary Fibrosis samples. Finally, we propose phenomenological scores of lung fibrosis and we show that they unambiguously sort out control and treated mice, with a better sensitivity and reproducibility in the subpleural region. These results should be readily generalized to other organs, as an accurate method to assess extracellular matrix remodeling during fibrosis.     

The morphology of the inner ear from the domestic pig (Sus scrofa)

The morphology of the hair cells of the inner ear end organs from the domestic pig ( Sus scrofa ) have been studied using a combination of Scanning and Transmission Electron Microscopy (SEM and TEM), revealing hair cells from the cochlea and vestibule using a novel surgical and technical approach. This is the first time that the inner ear hair cells from S. scrofa have been studied, thus providing useful anatomical information on the morphology of the hair cells from the cochlea, saccule and utricle from a large mammal. Anatomical information in relation to the morphology of the inner ear is of considerable importance, both in the pathological diagnosis of trauma and in the development of cochlea implants and other biotechnological systems associated with the enhancement of hearing. Standard fixation protocols using cardiac perfusion was not employed in this study as this method cannot always be applied, such as the pathological examination of the human ear, or the study of animals protected by endangered species legislation. With the exception of a very few countries, cetaceans cannot be killed for research purposes, yet physiological information on the inner ear from these species is urgently required for ecological assessment reasons. Supporting the use of S. scrofa as a model for cetacean hearing research is that this animal is a member of the order Artiodactyla, which includes both the hippopotamus and cetaceans. Being of a similar size, the pig is an ideal subject for developing protocols and surgical techniques required to investigate both the human and small cetacean auditory systems.     

Quantitative tomography of early mouse embryos: laser scanning microscopy and 3D reconstruction

The cell volume alteration participates in a wide variety of cellular functions that may interfere with intra‐cellular homeostasis. The most adequate approach of estimation of the volume changes induced by osmotic misbalance, alteration in shape and size due to the action shape forming substances, etc., is the direct measurement of volumetric parameters of embryos. In the given research, the volume magnitude and kinetics of changes in volume and surface area of blastomere and polar bodies of early mouse embryos were determined using three‐dimensional reconstruction of the optical section stack obtained with laser scanning microscope (LSM). The size and surface area were determined for isotonic and anisosmotic conditions. The physiological significance of the findings is discussed.     

Multipoint scanning dual‐detection confocal microscopy for fast 3D volumetric measurement

We propose a multipoint scanning dual‐detection confocal microscopy (MS‐DDCM) system for fast 3D volumetric measurements. Unlike conventional confocal microscopy, MS‐DDCM can accomplish surface profiling without axial scanning. Also, to rapidly obtain 2D images, the MS‐DDCM employs a multipoint scanning technique, with a digital micromirror device used to produce arrays of effective pinholes, which are then scanned. The MS‐DDCM is composed of two CCDs: one collects the conjugate images and the other collects nonconjugate images. The ratio of the axial response curves, measured by the two detectors, provides a linear relationship between the height of the sample surface and the ratio of the intensity signals. Furthermore, the difference between the two images results in enhanced contrast. The normalising effect of the MS‐DDCM provides accurate sample heights, even when the reflectance distribution of the surface varies. Experimental results confirmed that the MS‐DDCM achieved high‐speed surface profiling with improved image contrast capability.     

生物体内部结构三维显微成像技术研究

概述了生物体内部结构的几种传统的成像技术 ,介绍了一种新的生物体内部结构三维显微成像方法。该方法对生物体作连续切片 ,由 CCD显微摄像系统获取切片的序列图像 ,然后由计算机进行三维图像重构 ,最终得到生物体内部结构的三维显微图像     

Three-dimensional reconstruction of microstructures in gibbsite using charge contrast images

An iterative process of serial sectioning and charge contrast imaging has been applied to gibbsite particles embedded in epoxy resin. The features observed in the two-dimensional sections have been reconstructed into three-dimensional (3-D) objects, so that the volumetric relationships in the microstructure are visualised. The 3-D objects confirm that the image detail present in charge contrast images of gibbsite is related to the microstructure and the processes occurring during the crystallisation of gibbsite. Speculative explanations for what these microstructures may represent are presented.     

Knife-edge scanning microscopy for imaging and reconstruction of three-dimensional anatomical structures of the mouse brain

Anatomical information at the cellular level is important in many fields, including organ systems development, computational biology and informatics. Creating data sets at resolutions that provide enough detail to reconstruct cellular structures across tissue volumes from 1 to 100 mm³ has proven to be difficult and time-consuming. In this paper, we describe a new method for staining and imaging large volumes of tissue at sub-micron resolutions. Serial sections are cut using an automated ultra-microtome, whereas concurrently each section is imaged through a light microscope with a high-speed line-scan camera. This technique, knife-edge scanning microscopy, allows us to view and record large volumes of tissue in a relatively small amount of time (approximately 7 mm² s⁻¹).
  The resolution and scanning speed of knife-edge scanning microscopy provides a new method for imaging tissue at sufficient resolution to reconstruct maps of cellular distribution and morphology. We show that these techniques preserve the alignment of serial sections accurately enough to allow for reconstruction of neuronal processes and microvasculature. Expanding these techniques to other tissues opens up the possibility of creating fully reconstructed cellular maps of entire organs.     

豚鼠耳蜗Ca2+-ATP酶活性的超微细胞化学定位

In contrast to the perilymph,cochlear endolymph shows the special ionic components of a high K+ and low Na+.Bosher et al reported that the cochlear endolymph of laboratory rats contains very low Ca2+.     

Three-dimensional reconstruction of aqueous channels in human trabecular meshwork using light microscopy and confocal microscopy

Conventional two-dimensional imaging of the trabecular meshwork (TM) provides limited information about the size, shape, and interconnection of the aqueous channels within the meshwork. Understanding the three-dimensional (3-D) relationships of the channels within this tissue may give insight into its normal function and possible changes present in the eye disease glaucoma. The purpose of our study was to compare laser scanning confocal microscopy with standard 1 μm Araldite-embeddedhistologic sections for 3-D analysis of the trabecular meshwork. In addition, the study was done to determine whether computerized 3-D reconstruction could isolate the fluid spaces of the trabecular meshwork and determine the size of interconnections between the fluid spaces. Confocal microscopy appears comparable to 1 μm Araldite-embedded tissue sections and has the advantage of inherent registration of the serial tissue sections. Three-dimensional reconstruction allowed the isolation of the fluid spaces within the trabecular meshwork and revealed the presence of numerous interconnections between larger fluid spaces. The distribution of these interconnections was randomly arranged, with no predilection for specific regions within the trabecular meshwork. This distribution of constrictions and “expansion chambers” may provide a clue to the mechanism by which subtle histologic changes are associated with increased ocular pressure in glaucoma.     

A vision-based, 3D reconstruction technique for scanning electron microscopy: direct comparison with atomic force microscopy

High-resolution, detailed 3D reconstructions of biological specimens obtained from scanning electron microscopy stereo-micrographs and proprietary software were compared with Tapping-Mode AFM datasets of the same fields. The reconstruction software implements several original solutions including a neural adaptive point-matching technique, the ability to build an irregular triangulated mesh rather than a regular orthogonal grid, and the ability to re-map one of the original images exactly onto the reconstructed surface. The technique was applied to human nerve tissue to obtain 1,424 x 968-pixel, texture-mapped datasets, which were subsequently compared against 512 x 512-pixel AFM datasets from the same viewfields. Accounting for the inherent differences of the two techniques, direct comparison revealed an excellent visual match. The correspondence was also quantified by calculating the cross-correlation coefficient between corresponding altimetric profiles in SEM and AFM data, which consistently exceeded a figure of 0.9, with a rate of point mismatch in the order of 0.01%. Research is still underway to improve the robustness of the technique when applied to arbitrary images     

三维精密位移系统的设计

为满足精密位移的需要,研究开发了一种大行程、纳米级和计量型三维精密位移系统。采用模块化结构设计,即3个方向的驱动机构均采用完全相同的设计结构,分别称为X、Y、Z向一维工作台。位移系统在X、Y、Z3个方向采用粗、精两级驱动,并分别装有计量光栅。各方向粗驱动采用交流伺服电机配合精密丝杠和直线导轨进行,精驱动采用压电陶瓷微位移器配合柔性铰链进行,每个方向的两级驱动共用一套计量光栅,从而保证了位移系统的大行程、纳米级和计量型。介绍了位移系统的结构设计,分析了位移系统的位移分辨率、计量原理以及它的运动性能。实验表明,在40mm位移行程内,X、Y和Z向工作台两级驱动实际位移与设定位移之差分别不超过±0.030、±0.028和±0.033μm,验证了位移系统设计的有效性,为位移系统的设计提供了依据。     

Three-dimensional motion tracking for high-resolution optical microscopy, in vivo

When conducting optical imaging experiments, in vivo, the signal to noise ratio and effective spatial and temporal resolution is fundamentally limited by physiological motion of the tissue. A three-dimensional (3D) motion tracking scheme, using a multiphoton excitation microscope with a resonant galvanometer, (512 × 512 pixels at 33 frames s(-1)) is described to overcome physiological motion, in vivo. The use of commercially available graphical processing units permitted the rapid 3D cross-correlation of sequential volumes to detect displacements and adjust tissue position to track motions in near real-time. Motion phantom tests maintained micron resolution with displacement velocities of up to 200 μm min(-1), well within the drift observed in many biological tissues under physiologically relevant conditions. In vivo experiments on mouse skeletal muscle using the capillary vasculature with luminal dye as a displacement reference revealed an effective and robust method of tracking tissue motion to enable (1) signal averaging over time without compromising resolution, and (2) tracking of cellular regions during a physiological perturbation.     

基于二维工程图文件的Pro/E三维建模

由产品的三维模型可生成二维工程图,而由产品的二维工程图不能直接生成三维模型。给出了根据二维工程图快速建立三维模型的方法。     

Advanced spinning disk‐TIRF microscopy for faster imaging of the cell interior and the plasma membrane

Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high‐resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high‐resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD‐TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD‐TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup.     

Imaging and 3D morphological analysis of collagen fibrils

The recent booming of multiphoton imaging of collagen fibrils by means of second harmonic generation microscopy generates the need for the development and automation of quantitative methods for image analysis. Standard approaches sequentially analyse two-dimensional (2D) slices to gain knowledge on the spatial arrangement and dimension of the fibrils, whereas the reconstructed three-dimensional (3D) image yields better information about these characteristics. In this work, a 3D analysis method is proposed for second harmonic generation images of collagen fibrils, based on a recently developed 3D fibre quantification method. This analysis uses operators from mathematical morphology. The fibril structure is scanned with a directional distance transform. Inertia moments of the directional distances yield the main fibre orientation, corresponding to the main inertia axis. The collaboration of directional distances and fibre orientation delivers a geometrical estimate of the fibre radius. The results include local maps as well as global distribution of orientation and radius of the fibrils over the 3D image. They also bring a segmentation of the image into foreground and background, as well as a classification of the foreground pixels into the preferred orientations. This accurate determination of the spatial arrangement of the fibrils within a 3D data set will be most relevant in biomedical applications. It brings the possibility to monitor remodelling of collagen tissues upon a variety of injuries and to guide tissues engineering because biomimetic 3D organizations and density are requested for better integration of implants.     

液压扭矩板手的复杂三维问题的数值分析

运用三维非线性有限元分析方法，分析了一种液压扳手的三维应力和应变，得到了应力应变精确的数值解，发现了该结构的应力分布水平有较明显的不均匀，并探讨了圆角对扳手强度的影响，为进一步的优化设计提供了理论依据。     

High-resolution X-ray tomography of the human inner ear: synchrotron radiation-based study of nerve fibre bundles, membranes and ganglion cells

The combination of osmium tetroxide staining and high-resolution tomographic imaging using monochromatic X rays allows visualizing cellular structures of the human inner ear, that is, the organ of Corti, the stria vascularis and further soft tissues of the membranous labyrinth, in three-dimensional space with isotropic micrometre resolution. This approach permits to follow the course of nerve fibre bundles in a major part of the specimen and reveals the detailed three-dimensional arrangement of individual ganglion cells with distinct nuclei by means of X-ray tomography for the first time. The non-destructive neuron cell counting in a selected volume of 125 μm × 800 μm × 600 μm = 0.06 mm³ gives rise to the estimate that 2000 ganglion cells are present along 1 mm organ of Corti.     

彩色三维打印成形技术及应用

介绍了彩色三维打印(3DP)成形技术的工作原理及其成形工艺,分析了彩色三维打印的原型件的物理性能.此外,还介绍了彩色三维打印成形技术的应用.     

Multi-field 3D scanning light microscopy of early embryogenesis

A computer‐controlled microscopy system was devised to allow the observation of avian embryo development over an extended time period. Parallel experiments, as well as extended specimen volumes, can be recorded at cellular resolution using a three‐dimensional scanning procedure. The resulting large set of data is processed automatically into registered, focal‐ and positional‐drift corrected mosaic images, assembled as montages of adjacent microscopic fields. The configuration of the incubator and a sterile embryo chamber prevents condensation of the humidified culturing atmosphere in the optical path and is compatible with both differential interference contrast and epifluorescence optics. As a demonstration, recordings are presented showing the large‐scale remodelling of the embryonic primordial vascular structure.